• Journal of Life Science
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• v.27 no.12
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• pp.1479-1485
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• 2017

Muscle atrophy due to aging, starvation, and various chronic diseases leads to a decrease in muscle fiber area and density due to reduced muscle protein synthesis and increased protein breakdown. This study investigated the effect of dexamethasone and hydrogen peroxide on the induction of muscle atrophy and expression of atrophy-related genes in differentiated C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes in differentiation medium. During myoblast differentiation, muscle-specific transcription factors, such as myogenin, and MyoD expression increased. Differentiated C2C12 myotubes exposed to noncytotoxic levels of dexamethasone and hydrogen peroxide showed a decrease in myotube diameter, which was associated with up-regulation of muscle-specific ubiquitin ligases, such as muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and down-regulation of myogenin and MyoD. These results demonstrated that dexamethasone and hydrogen peroxide induced atrophy through regulation of muscle-specific ubiquitin ligases and muscle-specific transcription factors in C2C12 myotubes. In this study, we confirmed the process of differentiation of C2C12 myoblasts into myotubes in in vitro experiments in the presence of atrophy. This muscle atrophy model of C2C12 cells induced by dexamethasone or hydrogen peroxide seems suited to studies of the mechanism of muscle atrophy suppression and to exploit the experiment for excavating new muscle atrophy.

• BMB Reports
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• v.55 no.2
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• pp.104-109
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• 2022

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.3
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• pp.432-439
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• 2007

Thiazolidinediones (TZDs) act as potent activators of the adipose differentiation program in established preadipose cell lines. TZD's have also been investigated in diabetic patients and reported to act as PPAR-${\gamma}$ ligands. In this report, the effects of TZDs on the differentiation pathway of myoblasts have been investigated. C2C12 mouse myoblasts were grown in Dulbecco's Modified Eagles medium for 4-5 days until they reached almost 100% confluency. Post-confluent cells (day 0) were further exposed to adipogenic induction medium along with TZDs for 48 hours. Thereafter, cells were exposed only to TZDs every 48 h until day 10. The control was provided with differentiation medium without any treatment. Alterations in the cells during the differentiation programme were analyzed on the basis of fusion index, oil-red-o staining, adipocyte index, adipocyte stain uptake measurement, immuno-histochemistry and western blotting. Exposure of C2C12 mouse myoblasts to TZDs prevented the expression of myosin heavy chain with parallel increase in the expression of C/EBP-${\alpha}$ and PPAR-${\gamma}$ and acquisition of adipocyte morphology, thus abolishing the formation of multinucleated myotubes. TZDs exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes were insensitive to the compound. Continuous exposure (at least 4-5 doses) to inducers after the growth arrest was essential to provide a sustained environment to the cells converting to fully matured adipoctyes. The results indicate that TZDs specifically converted the differentiation pathway of myoblasts into that of adipoblasts.

• Journal of Life Science
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• v.17 no.7 s.87
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• pp.976-982
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• 2007

The purpose of this study was to determine the modulating effects of histone deacetylase inhibitor, trichostatin A, on the differentiation of mouse C2C12 myoblasts. We demonstrated that trichostatin A induced morphological changes of C2C12 myoblasts into smooth muscles and significantly increased the gene expression of smooth muscle markers including smooth muscle ${\alpha}-actin$ and transgelin. These results were due to the change in the expression level of cell cycle regulators in trichostatin A-treated C2C12 cells. Real-time PCR data revealed that cyclin dependent kinase inhibitor, p21, mRNA expression was significantly increased in trichostatin A-treated C2C12 cells. However, trichostaDn A rapidly decreased cyclin Dl mRNA expression necessary for cell cycle progression in 24hr after treatment. In conclusion, the strong inhibitory effects of trichostatin A on histone deacetylation induced transdifferentiation of C2C12 myoblasts into smooth muscle cells and these results are partly due to the changes in the expression of cell cycle regulators such as p21 and cyclin D1.

• Korean Journal of Clinical Laboratory Science
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• v.55 no.3
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• pp.184-194
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• 2023

In this study, we investigated whether p-anisaldehyde (PAA), the main component of essential oils derived from anise seeds, influences the differentiation of mouse C2C12 myoblasts. Cells were induced to differentiate over 5 days using a differentiation medium with or without PAA (50 or 200 mg/mL). Myotube length and diameter were measured, and the expressions of myogenic markers (myoblast determination protein 1, myogenin, myocyte enhancer factor 2, muscle creatine kinase, and myosin heavy chain) and atrophy-related genes (atrogin-1 and muscle ring finger-1 [MuRF-1]) were assessed by quantitative real-time polymerase chain reaction. Additionally, protein kinase B (Akt) phosphorylation was monitored by western blotting. PAA significantly induced the formation of smaller and thinner myotubes and reduced myogenic marker expression. Furthermore, PAA increased the expressions of atrogin-1 and MuRF-1 and simultaneously reduced Akt phosphorylation. Our findings indicate that PAA inhibits the myogenic differentiation of C2C12 cells by reducing the phosphorylation and activation of Akt.

• Biomedical Science Letters
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• v.29 no.1
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• pp.26-33
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• 2023

Ziziphus jujuba Mill. (ZJM), a traditional folk medicine and functional food in South Korea and China, has been reported to having pharmacological activities against anti-cancer, anti-oxidative, and anti-obesity. However, the effect of ZJM related to myoblast differentiation has not been known. In this study, we investigated the effects and mechanism of ZJM on myogenic differentiation of C2C12 cells. ZJM promotes myogenic differentiation and elevates the formation of multinucleated myotube compared to the control group. ZJM significantly increased the mRNA and protein expression of MyHC1, myogenin and MyoD in dose- and time-dependent manner. Interestingly, ZJM significantly inhibited the mRNA and protein expression of protein degradation markers, atrogin-1 and MuRF-1, in dose- and time-dependent manner. Taken together, our data suggest that ZJM is a potential functional candidate for muscle growth and strength by promoting myogenic differentiation.

• Journal of Korean Medicine for Obesity Research
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• v.23 no.2
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• pp.60-68
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• 2023

Objectives: This study was conducted to compare the effects of Hominis placenta (Jahage, J) and wild ginseng (SanSam, S) pharmacopuncture drugs on muscle differentiation and energy metabolism regulation in C2C12 myotubes. Methods: The C2C12 myoblasts were differentiated into myotubes for 5 days by replacing in medium containing 2% horse serum and then treated with J and S pharmacopuncture extract at different concentrations for 24 hr. The expression of myosin heavy chain and energy metabolism-regulating factors, myosin heavy chain (MHC), nuclear respiratory factor-1 (NRF-1), and proliferator-activated receptor γ coactivator-1 alpha (PGC-1α) were determined in C2C12 myotubes by western blot. Additionally, the phosphorylation of AMPK and the expression of mitochondrial biogenesis, including sirtuin 1 (SIRT1) were determined in the myotubes. Results: As a result, treatment with J and S pharmacopuncture extract at 0.1 and 1 mg/mL increased the MHC expression in C2C12 myotubes compared with non-treated cells, but only S pharmacopuncture was shown a significant and distinct increase in the expression. Expression of TFAM and NRF-1 was also shown significant increases in S and J pharmacopuncture in C2C12 myotubes compared to non-treated cells. The phosphorylation of AMPK and the expression of PGC-1α and SIRT1 showed increased expression in S and J pharmacopuncture compared to non-treated cells. The effect of low-dose of J pharmacopuncture on the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and PGC-1α expression was greater than that of S pharmacopuncture. Conclusions: In conclusion, both J and S pharmacopuncture promote muscle differentiation in C2C12 myoblasts into myotubes and energy metabolism through the AMPK/SIRT1 signaling pathway. This indicates that the pharmacopuncture with tonic herbal medicines can help to improve skeletal muscle function.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.162-168
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• 2020

Calcium is the most abundant stored mineral in the human body and is especially vital for bone health; thus, calcium deficiency can cause bone-related diseases, such as osteopenia and osteoporosis. However, a high concentration of serum calcium, which is commonly known as hypercalcemia, can also lead to weakened bones and, in severe cases, osteosarcoma. Therefore, it is necessary to maintain the concentration of calcium that is appropriate for bone biology. In the present study, we aimed to elucidate the effects of high concentration of calcium, approximately 2 folds the normal calcium level, on osteoblast differentiation. The CaCl2 treatment showed dose-dependent suppression of the alkaline phosphatase activity and mineralized nodule formation. Calcium showed cytotoxicity at an extremely high concentration, but a moderately high concentration of calcium that results in inhibitory effects to osteoblast differentiation showed no signs of cytotoxicity. We also confirmed that the CaCl2 treatment repressed the mRNA expression and protein abundance of various osteogenic genes and transcriptional factors. Considered together, these results indicate that a high concentration of calcium negatively regulates the osteoblast differentiation of C2C12 cells.

• Food Science and Preservation
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• v.31 no.2
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• pp.298-306
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• 2024

This study evaluated the differentiation and protective effects of mixed plant-extract powder in C2C12 muscle cells. Cells were differentiated into myotubes in 2% horse serum (HS)-containing medium with mixed plant-extract powder (MPEP) for 6 days. Treatment with MPEP increased the expression of myogenin and myosin heavy chain (MHC) protein in cells compared with non-treated cells. Differentiated cells were pretreated with MPEP, and hydrogen peroxide (H₂O₂). Our results revealed that treatment with MPEP before H₂O₂ treatment increased cell viability and decreased H₂O₂-induced lactate dehydrogenase (LDH) and creatine kinase (CK). In addition, MPEP attenuated H₂O₂-induced upregulation of Bax, downregulation of Bcl-2, and activation of caspase-9 and -3. These results suggest the MPEP can stimulate C2C12 muscle cell differentiation into myotubes and observe the protective effect of mixed plant-extract powder against muscle oxidative stress. In conclusion, MPEP may be useful as a prevention and treatment material for skeletal muscle disease caused by age-related diseases.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1115-1119
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• 2007

This study was conducted to determine the difference between satellite cells (porcine) and myoblasts (C2C12) in their differentiation under the influence of 2, 4-thiazolidindion. C2C12 myoblast cells and porcine satellite cells (isolated from 10 d old $Landrace{\times}Duroc$ piglets) were grown to absolute confluency. Post confluent cells (day 0) were further exposed to adipogenic induction medium along with 2, 4-thiazolidindion ($8{\mu}M$) for 2 d. Thereafter, cells were exposed to 2, 4-thiazolidindion alone every 2 d till day 10 and analysed. The control was cultured in differentiation medium without any treatment. Increased (p<0.05) expression of transcriptional factors i.e. C/EBP-${\alpha}$ and PPAR-${\gamma}$ and transition of cells to adipocyte morphology was noticed from 2 d and 4 d onwards in satellite cells (Porcine) and myoblasts (C2C12) respectively. Myogenesis was observed to be suppressed completely in case of satellite cells compared to myoblasts in response to 2, 4-thiazolidindion. Pax-7 (transcriptional factor) appeared as a sole entity to satellite cells only, as it was not identified in case of myoblasts. Although both the cells were converting to adipoblasts, the degree of their conversion was different in response to 2, 4-thiazolidindion. Therefore, the hypothesis that satellite cells contribute various domains to the growing myoblasts appeared obscured and found to be dependent on the proliferative energy/or degree of fusion. However, it revealed satellite cells as currency to myoblasts/muscle.